PROJECT SUMMARY Mechanical loading and pharmaceutical interventions both improve bone mechanical properties, but there is a critical gap in our understanding of the role that collagen plays in mediating these effects. This gap in knowledge by which collagen processing, organization, mineralization, and hydration change with combined load and drug treatment is a critical impediment to the development of combination therapies that increase fracture resistance by targeting tissue moieties other than mineral. Our long-term goal is to develop ways to alter physical properties of bone tissue to increase fracture resistance. The overall objective in this application is to elucidate how mechanical loading and a RAL-analog (RALA) modify newly forming and pre-existing bone to decrease fragility. The central hypothesis is that in addition to changes in mass and mineral, collagen-modifying effects exist for both loading and RALA, the combination of which interactively improve mechanical integrity beyond the effects of either monotherapy. The premise of this hypothesis stems from preliminary data generated in the applicants' laboratories. The rationale for the proposed work is that successfully making bone stronger and more resistant to fracture by combining RALA's hydrating effects with mechanical regulation of bone mass and perilacunar matrix activity could provide alternative ways for the orthopaedic community to approach the treatment of bone diseases. Guided by preliminary data, this hypothesis will be tested using three specific aims: 1) to define influences of loading on osteocyte perilacunar matrix activity and osteoblast matrix deposition; 2) to determine how RAL/RALA modify collagen quality and matrix hydration; and 3) to determine interactive effects of loading and RALA. Under the first aim, techniques already in place will be used to investigate in vitro and in vivo loading effects in healthy cells and animals, as well as in models of disrupted collagen synthesis. In vitro loading will be induced by substrate stretching for osteoblasts or pulsatile fluid flow for osteocytes. Gene expression of enzymes and chaperones will be quantified, as well as molecules associated with resorption. Matrix production, organization, composition and mechanical integrity will be assessed. For in vivo loading experiments, similar techniques will be used to assess collagen synthesis, post-translational modifications, and crosslinking along with nanoscale and whole bone tests of mechanical integrity, fatigue resistance and fracture toughness. In Aim 2, outcome measures from Aim 1 will be used to investigate the effects of RAL/RALA as a function of disease state. In Aim 3, interactive effects of combined loading and drug-based treatment will be assessed. The approach is innovative because of its focus on collagen, in addition to mass and architecture. It also focuses on osteoblast- produced collagen on surfaces and changes induced by osteocytes throughout the bone. This work is significant because it will demonstrate that interactions through combination therapies can improve skeletal mechanical phenotypes, not by correcting the disease cause, but by impacting collagen synthesis, assembly, mineralization, and tissue hydration. Such knowledge will provide new ways to approach treatment of fragility-related diseases.